Single cell and collective cell migration are fundamental processes that enable human development, immune responses and wound healing while also playing a key function in cancer progression. Our overall goal in this project is to understand the fundamental molecular and cellular mechanisms of how single cells and groups of human cells migrate. We will be focusing on two in vitro cell models, human umbilical vasculature endothelial cells (HUVEC) to study collective directed migration in response to growth factor and a cell model for neutrophils, differentiated HL-60 cells, to investigate single cell chemotaxis. These model systems were chosen since they allow us to use automated imaging of important functional migration parameters to explore the role of a large numbers of migration related genes using siRNAs knockdown and to perform a phenotypic classification. Our project is taking a systems approach, using small interference RNAs to perturb different parts of the cell migration machinery as well as computational modeling approaches. We are also employing rapid chemical perturbation methods of the PI3K and other pathways that our laboratory developed as well as high resolution fluorescent imaging using biosensors and markers for cell migration. By perturbing and monitoring local signaling events, we will be exploring different hypotheses of how cells polarize, steer their front and migrate with the goal to generate a quantitative molecular and mechanistic model for directed migration in these cell models. Insights into new regulators of migration and the roles that different regulators play in the overall endothelial and leukocyte migration processes will likely lead to the identification of new drug targets relevant for vasculature, immune diseases or cancer. PUBLIC HEALTH RELEVANCE: Single cell and collective cell migration are fundamental processes that enable human development, immune responses and wound healing while also playing a necessary function in cancer progression. Our overall goal in this grant is to understand the key molecular and cellular mechanisms of how single cells and groups of human cells migrate.